Plant Infection Method Research Articles

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

A new sand pouch-plant infection technique for enumeration of rhizobia in soil

A sand pouch-plant infection technique for counting most probable numbers of rhizobia in soil is described. Populations of <em>Rhizobium leguminosarum</em> bv. <em>trifoli</em> detected by the plant infection method performed in enclosed glass tubes or in sand pouches did not differ significantly. The described method was used to assess numbers of <em>R. leguminosarum</em> bv. <em>trifoli</em> (<em>R.l.t</em>.) and bv. viciae (<em>R.l.v.</em>) in 20 soils of Poland. Logarithms of the populations of <em>R.l.t.</em> in the tested soils ranged from not detectable level to 4.76 and those of <em>R.l.v.</em> from 2.23 to 5.84 in g1 of soil dry mass. Numbers of <em>R.l.t.</em> were significantly correlated with soil clay, C org, and total N contents but not with the soil pH (in KCI), while numbers of <em>R.l.v.</em> showed significant correlation only with the soil pH.

Symbiotic and phenotypic diversity of Rhizobium leguminosarum bv. viciae from Northern Gondar, Ethiopia

Rhizobia that nodulate cool season legumes are widely spread in the Mediterranean and highland agro ecosystems. Faba bean is one of these important crops that represent the major protein source of human nutrition, cash crop for farmers and component of the farming systems. This study is aimed at identifying rhizobia isolates capable of guarantying efficient nitrogen derived from biological nitrogen fixation. Due to this, a total of twenty-one isolates of Rhizobium were isolated from as many sampling sites of northern Gondar using plant infection method. The isolates were characterized morphologically and physiologically and tested on sand and soils to evaluate their symbiotic effectiveness. Studies on symbiotic effectiveness on sand culture indicated that, the strains showed shoot dry matter ranging from 0.4 (AUFR127) to 2.3 g/plant (AUFR124), with negative control (0.2 g/plant) and positive control of 2.4 g/plant. Eighty percent of the isolates were found to be effective and have very effective nitrogen fixers based on their shoot dry matter accumulation (50 to 100%) in relation to the nitrogen fertilized control plants. Culturally, almost all of them displayed large colonies with diameters of 2 to 4.5 mm, generation time of 1.9 to 4.3 h and showed characteristics of fast growing rhizobia. With a few exceptions, isolates grew at temperatures of 15 and 35°C and were found to be sensitive to salt, except AUFR118 that grew up to 5% NaCl. The isolates also grew on a wide range of moderate acidity and alkalinity (pH5.5-9).With the exception of gluconate, citrate and tartarate, almost all isolates grew on all carbohydrates. The pattern of intrinsic antibiotic resistance showed that, almost all of them were tolerant to chloramphenicol, nalidixic acid and erythromycin. Isolates AUFR118, AUFR128 and AUFR132 were found to be resistant to almost all tested antibiotics. These isolates were also included in the effective and very effective symbiotic groups. The trial of the 5 selected effective isolates on two soil types showed that, they performed well without any significant difference on their respective soils. Interestingly, the isolates were found to nodulate the host on the highly acidic soil (pH 4.8) that failed to be nodulated by the indigenous rhizobia. Generally, the present work shows the physiological and symbiotic diversity of the isolates in the traditional agricultural areas of the region and the potential of these rhizobia to be used as effective commercial inoculants in areas where the indigenous rhizobia fail to do so. This will help to reclaim acidic soils for faba bean production. Key words : Faba bean, Northern Gondar, Rhizobium, phenotypic diversity, symbiotic effectiveness

Symbiotic and phenotypic diversity of Rhizobium leguminosarum bv. viciae from Northern Gondar, Ethiopia

Rhizobia that nodulate cool season legumes are widely spread in the Mediterranean and highland agro ecosystems. Faba bean is one of these important crops that represent the major protein source of human nutrition, cash crop for farmers and component of the farming systems. This study is aimed at identifying rhizobia isolates capable of guarantying efficient nitrogen derived from biological nitrogen fixation. Due to this, a total of twenty-one isolates of Rhizobium were isolated from as many sampling sites of northern Gondar using plant infection method. The isolates were characterized morphologically and physiologically and tested on sand and soils to evaluate their symbiotic effectiveness. Studies on symbiotic effectiveness on sand culture indicated that, the strains showed shoot dry matter ranging from 0.4 (AUFR127) to 2.3 g/plant (AUFR124), with negative control (0.2 g/plant) and positive control of 2.4 g/plant. Eighty percent of the isolates were found to be effective and have very effective nitrogen fixers based on their shoot dry matter accumulation (50 to 100%) in relation to the nitrogen fertilized control plants. Culturally, almost all of them displayed large colonies with diameters of 2 to 4.5 mm, generation time of 1.9 to 4.3 h and showed characteristics of fast growing rhizobia. With a few exceptions, isolates grew at temperatures of 15 and 35°C and were found to be sensitive to salt, except AUFR118 that grew up to 5% NaCl. The isolates also grew on a wide range of moderate acidity and alkalinity (pH5.5-9).With the exception of gluconate, citrate and tartarate, almost all isolates grew on all carbohydrates. The pattern of intrinsic antibiotic resistance showed that, almost all of them were tolerant to chloramphenicol, nalidixic acid and erythromycin. Isolates AUFR118, AUFR128 and AUFR132 were found to be resistant to almost all tested antibiotics. These isolates were also included in the effective and very effective symbiotic groups. The trial of the 5 selected effective isolates on two soil types showed that, they performed well without any significant difference on their respective soils. Interestingly, the isolates were found to nodulate the host on the highly acidic soil (pH 4.8) that failed to be nodulated by the indigenous rhizobia. Generally, the present work shows the physiological and symbiotic diversity of the isolates in the traditional agricultural areas of the region and the potential of these rhizobia to be used as effective commercial inoculants in areas where the indigenous rhizobia fail to do so. This will help to reclaim acidic soils for faba bean production. Key words: Faba bean, Northern Gondar, Rhizobium, phenotypic diversity, symbiotic effectiveness.

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

Abstract The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10 4 (ANU) and 4.2 × 10 3 (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.

Populations of rhizobia in some Polish soils not planted with legumes

Using the soil dilution – plant infection method, populations of several species of root-nodule bacteria (rhizobia) were enumerated in 46 soil samples collected throughout Poland. To exclude effects of host-plant cultivation on the interactions between soil physico-chemical properties and populations of the rhizobia, only soils not planted with legumes during at least the last 10 years were analysed. Rhizobium leguminosarum bv. viciae (symbionts of pea, faba bean, vetch) and R. leguminosarum bv. trifolii (symbionts of clover) were detected in 44 and 42 soils, respectively. Most of these soils contained moderate and high numbers of these species rhizobia. Symbionts of beans, R. leguminosarum bv. phaseoli, and symbionts of lupine, Bradyrhizobium sp. (Lupinus) were less frequent in the examined soils, of which 11 contained non-detectable populations of these bacteria. Sinorhizobium meliloti, rhizobia nodulating alfalfa, were sparse in the test soils, with 32 soil samples containing no detectable numbers of S. meliloti and only 3 samples harbouring moderate populations of this species. The estimated numbers of rhizobia were also related to the following properties of the soils: organic C and total N contents, pH in water and in KCl, and the content of soil mechanical fractions <0.02 mm and <0.002 mm. Results of this study have shown that soil texture (particularly clay content) and soil reaction have the greatest influence on populations rhizobia in soils.

Enrichment of Bradyrhizobium spp populations in soil due to cropping of the homologous host legume

Host legumes can enrich their immediate soil environment with rhizobia through rhizosphere effects. The extent to which this enrichment occurs, the specificity of the process and its interaction with soil management factors remain poorly described. In a series of field trials, we measured changes in the size of indigenous populations of Bradyrhizobium in response to cropping of host and non-host legumes under two N fertilizer regimes. Uninoculated cowpea ( Vigna unguiculata) and soybean ( Glycine max) were grown with or without applied urea (900 kg N ha −1) at three field sites on the island of Maui, HI, U.S.A., not previously cropped with legumes. Using the most-probable-number plant infection method and Siratro ( Macroptilium atropurpureum) as the host, the population density of Bradyrhizobium in the bulk soil at each site was measured at planting and at grain maturity and compared to the population density in adjacent fallow soil. When the size of the initial indigenous population was low (18 and 580 cells g −1 soil), significant increases in the population density compared to fallow soil were observed only in soils cropped with cowpea receiving no applied urea. When the size of the initial indigenous bradyrhizobial population was high (5.8 × 10 4 cells g −1 soil), no significant increase in the population density was observed. These results suggested that enrichment of soil bradyrhizobial populations was host-specific, that symbiotic legumes can enrich their soil environment with microsymbionts up to a threshold level and that such enrichment can be curtailed by soil management practices that suppress nodulation.

Counting Rhizobium sp. ( Phaseolus) by a plant-infection method using a small-seeded cultivar of Phaseolus silvestre L. grown enclosed in test tubes

We report laboratory experiments that tested the suitability of a wild-type small-seeded cultivar of Phaseolus silvestre L., Morelos 628, for counting Rhizobium sp. ( Phaseolus) by the most probable number method (MPN). There was good agreement between MPN estimates of numbers in pure cultures using Morelos 628 grown in test tubes and a larger commercial variety of P. vulgaris L. grown in Leonard jars, and with colony counts on yeast-mannitol agar (YMA) plates. Morelos 628 did not nodulate in bacteria-free conditions when inoculated with unrelated strains of root nodule bacteria. After 28 days, MPN estimates of R. sp. ( Phaseolus) were not significantly different from counts made after 3 days on YMA, at either low or high cell concentrations in pure culture or in unsterile soil. Nodulation of Morelos 628 was at a maximum after 21 days.

Semienclosed Tube Cultures of Bean Plants ( Phaseolus vulgaris L.) for Enumeration of Rhizobium phaseoli by the Most-Probable-Number Technique

The semienclosed tube culture technique of Gibson was modified to permit growth of common bean (Phaseolus vulgaris L.) roots in humid air, enabling enumeration of the homologous (nodule forming) symbiont, Rhizobium phaseoli, by the most-probable-number plant infection method. A bean genotype with improved nodulation characteristics was used as the plant host. This method of enumeration was accurate when tubes were scored 3 weeks after inoculation with several R. phaseoli strains diluted from aqueous suspensions, peat-based inoculants, or soil. A comparison of population sizes obtained by most-probable-number tube cultures and plate counts indicated that 1 to 3 viable cells of R. phaseoli were a sufficient inoculant to induce nodule formation.

Fractionation and Estimation of Particle-Attached and Unattached Bradyrhizobium japonicum Strains in Soils

Rhizobial cells attached or unattached to soil particles were estimated. Nonsterile soils into which antibiotic-resistant mutants of Bradyrhizobium japonicum had been introduced were fractionated by a centrifugation technique into two fractions: A, which contained mainly rhizobial cells attached to soil particles, and F, which contained mainly rhizobial cells unattached to them. Rhizobial counts decreased in both fractions during incubation of the soil at 30 degrees C, with a concomitant decrease in the proportion of the count of fraction F to that of fraction A. Sonication of fraction A of the soil incubated for more than 3 weeks caused an increase in the rhizobial count. The ratio of the count of fraction A estimated by the plant infection method to that estimated by the dilution plate method increased after 5 days of soil incubation. More than 90% of the indigenous rhizobia in an agricultural field existed in fraction A. These results suggest that the majority of rhizobial cells are attached to soil particles.

Cowpea rhizobia: Comparison of plant infection and plate counts

Enumeration of Rhizobium in soil is usually accomplished by the most probable number (MPN) plant infection method. The accuracy of MPN counts as compared to plate counts was determined for five strains of cowpea rhizobia. Host plants included cowpea ( Vigna unguiculata (L.) Walp.), siratro ( Macroptilium atropurpureum (DC.) Urb.) and peanut ( Arachis hypogaea L.). Plastic growth pouches were used primarily for cowpea and siratro while plastic cups containing vermiculite were used for peanut. The number of rhizobia determined by the MPN method using cowpea, siratro and peanut underestimated the population from 10- to 100-fold. A control experiment using soybeans ( Glycine max (L.) Merrill) indicated that the MPN method was accurate for R. japonicum. Experimentors using the MPN method should be aware of its accuracy for particular Rhizobiutn-legume combinations.